The structure and genetic coding sequences of a family of developmental proteins in plants have homology to mammalian QM proteins and to genes encoding the proteins. Recombinant molecules comprising plant QM coding regions and suitable promoters, are used to produce a transformed plant with altered development. The altered development causes male sterility.
The expression of most, if not all, plant genes can be considered to be related in some way to plant development. Many classes of genes are known to respond to development signals involved in cell differentiation, formation of tissues and organs, or in controlling plant growth. There are several well-characterized examples: genes that are regulated by light (such as rbcS and cab gene families), or by hormones and genes that express specifically in anthers, roots, seeds or leaves, or in specific cell types in these tissues (See Edwards and Coruzzi, 1990 and Kuhlmeier, Green and Chua, 1987 for reviews). Other types of genes are known to regulate the expression of yet other genes, such as the maize regulatory gene Opaque2 that codes for a transcriptional activator which regulates the expression of 22kd zein genes (Schmidt et al., 1992, Ueda et al., 1992) or the C1 and R genes in maize that code for transcriptional activators that regulate the expression of A1 and BZ1 (Klein et al., 1989).
A very new area of research in plants includes the identification and isolation of genes from plants, which, based on their homology to genes from animal and yeast systems, are believed to be involved in the control of basic cell processes such as cell division (See Jacobs, 1992 for a review). An example of such a gene is the homologue of the yeast cdc2 gene which has been cloned from maize (Colasanti, et al., 1991). In the future, there are certain to be additional genes identified in plants which control other basic cellular or developmental processes.
In mammals, developmental proteins have been implicated in abnormal cell division such as characterizes the malignant state. For example, Wilms' tumor is a pediatric tumor of the kidney which arises in embryonic blastoma cells and occurs in both sporadic and hereditary forms. Three groups have reported the cloning of two distinct genes which are associated with Wilms' tumor. The first, WT1, encodes a zinc finger protein belonging to the early growth response (EGR) gene family and maps to the llp13 locus in humans, which is often deleted in tumorigenic cells (Call et al., 1990, Gessler et al., 1990). The second gene, termed QM, was originally cloned by Dowdy et al., Nuc. Acids Res., 19: 5763-5769, (1991) through the use of subtractive hybridization using cDNAs and RNA derived from tumorigenic and non-tumorigenic Wilms' microcell hybrid cells, respectively. This gene was shown to be expressed at the RNA level in virtually all normal tissues examined in the mouse but was lacking in Wilms' tumorigenic cell lines.
The protein encoded by this gene is 25 kD in size and is very basic with a pI of approximately 12.0. Dowdy also demonstrated that QM is a member of a family of genes in a number of mammals, particular primates. van den Ouweland et al. (1992) cloned the QM gene from a human Xqter chromosome library and showed that this gene was 100% similar to the previously cloned QM gene. The expression of the QM gene has been demonstrated in the mouse (Dowdy et al., 1991), has been cloned in the chicken, and with data from van den Ouweland et al., suggests that this gene is conserved across a large phylogenetic range. It was postulated that QM may be involved in maintenance of the non-tumorigenic phenotype (Dowdy et al. 1991). It would not be expected to find the QM gene in plants, which do not have comparable phenotypes.
Discovery of genes which would alter plant development would be particularly useful in developing genetic methods to induce male sterility because other methods currently available have serious shortcomings (e.g., detasseling, CMS, SI, and the like).
Production of hybrid seed for commercial sale is a large industry. Hybrid plants grown from hybrid seed benefit from the heterotic effects of crossing two genetically distinct breeding lines. The agronomic performance of this offspring is superior to both parents, typically in vigor, yield and uniformity. The better performance of hybrid seed varieties compared to open-pollinated varieties makes the hybrid seed more attractive for farmers to plant and thereby commands a premium price in the market.
In order to produce hybrid seed uncontaminated with self-seed, pollination control methods must be implemented to ensure cross-pollination and not self-pollination. Pollination control mechanisms can be mechanical, chemical or genetic.
A mechanical method for hybrid seed production can be used if the plant species in questions has spatially separate male and female flowers or separate male and female plants. The corn plant, for example, has pollen producing male flowers in an inflorescence at the apex of the plant and female flowers in the axils of leaves along the stem. Outcrossing is assured by mechanically detasselling the female parent to prevent selfing. Even though detasseling is currently used in hybrid seed production, the process is labor intensive and costly (yield loss is incurred).
Most major crop plants of interest, however, have both functional male and female organs within the same flower so emasculation is not a simple procedure. It is possible to remove by hand the pollen forming organs before pollen shed, however, this form of hybrid seed production is extremely labor intensive and, hence, expensive. Seed is produced in this manner if the value and amount of seed recovered warrants the effort.
A second general method of producing hybrid seed is to use chemicals that kill or block viable pollen formation. These chemicals, termed gametocides, are used to impart a transitory male-sterility. Commercial production of hybrid seed by use of gametocides is limited by the expense and availability of the chemicals and the reliability and length of action of the applications. These chemicals are not effective for crops with the extended flowering period because new flowers will be produced that will not be affected. Repeated application of chemicals is impractical because of costs.
Many current commercial hybrid seed production systems for field crops rely on a genetic method of pollination control. Plants that are used as females either fail to make pollen, fail to shed pollen or produce pollen that is biochemically unable to effect self-fertilization. Plants that are unable (by several different means) to self pollinate biochemically are termed self-incompatible. Difficulties associated with the use of self-incompatibilities include availability and propagation of the self-incompatible female line and stability of the self-compatibility. In some instances, self-incompatibility can be overcome chemically or immature buds can be pollinated by hand before the biochemical mechanism that blocks pollen is activated. Self-incompatible systems that can be deactivated are often very vulnerable to stressful climatic conditions that break or reduce the effectiveness of the biochemical block to self-pollination.
Of more widespread interest for commercial seed production are systems of pollen control based genetic mechanisms causing male sterility. These systems are of two general types: (a) genic male sterility, which is the failure of pollen formation because of one or more nuclear genes or (b) cytoplasmic-genetic male sterility (commonly called cytoplasmic male sterility or CMS) in which pollen formation is blocked or aborted because of a defect in a cytoplasmic organelle (mitochondrion).
Nuclear (genic) sterility can be either dominant or recessive. A dominant sterility can only be used for hybrid seed information if propagation of the female line is possible (for example, via in vitro clonal propagation). A recessive sterility could be used if sterile and fertile plants are easily discriminated. Commercial utility of genic sterility systems is limited however by the expense of clonal propagation and roguing the female rows of self-fertile plants.
Many successful hybridization schemes involve the use of CMS. In these systems, a specific mutation in the cytoplasmically located mitochondrion can when in the proper nuclear background, lead to the failure of mature pollen formation. In some instances, the nuclear background can compensate for the cytoplasmic mutation and normal pollen formation occurs. The nuclear trait that allows pollen formation in plants with CMS mitochondria is called restoration and is the property of specific "restorer genes". Generally, the use of CMS for commercial seed production involves the use of three breeding lines, the male-sterile line (female parent), a maintainer line which is isogenic to the male-sterile line but contains fully functional mitochondria and the male parent line.
The male parent line may carry the specific restorer genes (usually designated a restorer line) which then imparts fertility to the hybrid seed. For crops, such as vegetable crops for which seed recovery from the hybrid is unimportant, a CMS system could be used without restoration. For crops for which the fruit or seed of the hybrid is the commercial product then the fertility of the hybrid seed must be restored by specific restorer genes in the male parent or the male-sterile hybrid must be pollinated. Pollination of non-restored hybrids can be achieved by including with hybrids a small percentage of male fertile plants to effect pollination. In most species, the CMS trait is inherited maternally (because all cytoplasmic organelles are inherited from the egg cell only), which can restrict the use of the system. Although still used for a number of crops, limitations of CMS systems have a tendency to break down with prolonged use. Generally, male sterility is less than 100% effective. One particular CMS type in corn (T-cytoplasm) confers sensitivity to infection by a particular fungus.
A search for methods of altering development in plants, for example, to produce male sterile plants, revealed an exceptionally suitable family of developmental proteins.